Lecture 9 - DNA Replication and Repair Flashcards
What are and explain the 3 proposed models of how DNA replication works
1) Conservative - the two parental DNA strands would re-basepair with each other after being used as templates to synthesize new strands, same with the two new strands being together
2) Semi-conservative - each of the two parental DNA strands would act as a template for new DNA strands to be synthesized, but after replication, each parental DNA strand would basepair with the complementary newly-synthesized strand, and both double-stranded DNAs would include one parental or “old” strand and one daughter or “new” strand
3) Dispersive - after replication both copies of the new DNAs would somehow have alternating segments of parental DNA and newly-synthesized DNA on each of their two strands
Describe bacterial DNA replication
Begins at replication origin (replication forks), proceeds bidirectionally (theta replication), once finished, the linked circles are separated by topoisomerase, new cell walls are built
Explain the differences between DNA replication in bacteria vs humans
Humans are slower at replicating but have so much more DNA, due to this we have many more points of origin to replicate more DNA
Describe eukaryotic replication
It has multiple sites of origin, multiple replication units (replicons), each replicon contains an origin of replication
Describe briefly the steps of eukaryotic DNA replication
Replication begins and a point of origin, replication bubbles form, and then the bubbles/replicons fuse together to form four linear strands (2 old/2 new)
Describe pre replication in prokaryotes
DnaA protein binds to the 9-mer region, forcing unwinding of the 13-mer region, then DnaC brings DnaB to the 13-mer region to initiate helicase activity
Describe the pre-replication complex in eukaryotes
Origin recognition complex (ORC) binds to replication origin (multi-subunit protein complex), then minichromosome maintenance (MCM) proteins bind, these contain helicases that unwind DNA, and helicase loader proteins bind and help MCM bind to the ORC
How is licensing regulated?
Cdk’s (protein kinases) are activated during early S phase and active DNA synthesis at licensed origins and prevents origins from being licensed again, they phosphorylate ORC and helicase loader protiens
Geminin blocks the binding of MCM proteins to DNA, after cell division, geminin is degraded and Cdk is inhibited
What do DNA polymerases do?
Add new nucleotides complementary to the template strand at the 3’ OH of the growing strand
What do primases do?
Add short RNA primers to form short RNA/DNA duplexes
What do topoisomerases do?
Reduces supercoiling and prevents twisting of DNA
What do helicases do?
Unwind the DNA
What do single strand binding proteins (SSB’s) do?
Binds DNA to keep in unwound
Which way does DNA polymerase synthesize DNA in?
5’ - 3’
5 in prokaryotes
12 in eukaryotes
What happens during DNA synthesis?
Phosphodiester bond if formed between 3’ OH and 5’ phosphate
PPi is released, this provides energy for synthesis
What are okazaki fragments and why do they occur?
Short pieces of newly synthesized DNA on the lagging strand, they happen because DNA polymerase only works in the 5’-3’ end, so it needs to move backwards essentially and make it in pieces
Name and give the function of the 2 RNA primers in DNA replication
DNA polymerase III adds nucleotides to the 3’ end of an existing nucleotide chain
DNA polymerase I has a 5’-3’ exonuclease activity to remove RNA and fill in gap when DNA reaches an RNA primer
Steps in DNA replication with all the components
Initiator protein binds to dsDNA (slight unwinding), DNA helicase (continues unwinding), DNA gyrase (relaxes supercoils), SSB (stabilizes single strands), Primase binds (synthesizes RNA primer), DNA polymerase III (adds nucleotides 5’-3’ direction, RNA primer made for lagging strand, DNA polymerase III extends), Discontinuous synthesis of lagging strand (multiple primers and okazaki fragments), DNA polymerase I (removes RNA primers), then DNA ligase (links okazaki fragments)
What is the replisome?
Proteins involved in DNA replication are all closely associated in one large complex
What additional proteins do eukaryotes have compared to prokaryotes?
Replication factories synthesize DNA as chromatin fibers are fed through them
Chromatin remodeling proteins loosen nucleosome packing ahead of the replication fork
What is the problem at the end of replication?
There is no 3’OH to add nucleotides to at the end of linear DNA. It cannot properly replicate and there is not enough room for another okazaki fragment so the DNA strand gets shortened
What is the mechanism for protecting the shortening of DNA?
Telomeres (protective buffer) - is a highly repeated DNA sequence (TTAGGG), its non-coding, this ensures that any loss will not be from an important sequence
What is the enzyme called that synthesizes telomeres?
Telomerase - its an RNA/protein hybrid, its not active in all cells, it acts as a template to create more telomeres, also recruits capping proteins to protect the end of the telomere
What is the function of telomerase?
RNA matches telomere repeat, its positioned so the enzyme can extend the 3’ end
It adds more copies to the top (longer) strand and makes room for another okazki fragment on the lower (shorter) strand
